This invention relates to bioreactors for mass culture of mammalian cells, and more particularly to a bioreactor system having a multitubular array which simulates in vivo capillary bed conditions wherein anchorage-dependent and anchorage-independent mammalian cells can be cultured.
New technologies for creating biologically active molecules, including monoclonal antibodies, have created a vigorous biotechnology industry. While new methods for introducing genes into cells to make desired biomolecules are being announced on a weekly basis, the problems of producing the biomolecules on a mass scale have not been solved. Existing microbial fermentation systems are often of little use in producing many of the currently desired biomolecules largely because bacteria produce proteins which are different from those of mammalian cells. As a consequence, mammalian cell growth is becoming a very important area in the production of pharmaceutical products. Unfortunately, all currently available bioreactors used for growth of mammalian cells suffer from one or more of the following problems: inadequacy of oxygen transfer, accumulation of toxic wastes, inability to be scaled up, and difficulty in automation.
In vivo production of monoclonal antibodies, for example, involves the intraperitoneal inoculation of mice with antibody-producing hybridoma cells followed by collection of ascites which are fluid-rich in antibodies. However, one mouse often takes up to 8 weeks to produce 40-200 mg antibody and the recovered antibodies are contaminated with mouse proteins that can cause serum sickness when injected into humans. Moreover, mice are expensive to maintain and the cost of production does not decrease with scale-up. Additionally, human monoclonal antibodies cannot be cultured in mice.
In view of the above-stated disadvantages, in vitro culturing techniques, which generally are categorized as either batch or continuous systems, are preferred. However, cell densities in vitro usually do not exceed 1.times.10.sup.7 cells/ml and the resulting concentrations of product are also low (0.01-0.1 mg/ml vs. 1-10 mg/ml in vivo).
Batch culture involves growth in a batch of medium with no attempt to separate medium and product from cells continuously. A major problem with batch culture systems is poor oxygen transfer. Mammalian cell oxygen demand ranges between 0.053 mmole O.sub.2 /L-hr-10.sup.6 cells/ml to 0.59 mmole O.sub.2 /L-hr-10.sup.6 cells/ml. Unlike microbial fermentations, bubbles of oxygen cannot be forced through the reactor since mammalian cells lack rigid cell walls and often cannot withstand the resulting shear forces. Several suggested approaches for aerating the cells, such as use of airlift convection forces and restricted impeller techniques, have been employed. However, these approaches only reduce the shear forces and physical trauma to the cells, but do not eliminate these problems. Nevertheless, cell yield is still limited by oxygen concentration. A further significant problem is that of waste elimination. Centrifugation can be used to remove old medium, but this increases the probability of contamination and can also result in substantial cell damage.
Encapsulation of cells either in an alginate gel or in alginate gel surrounded by a semipermeable polymer membrane with controlled pores is another method for cultivating mammalian cells. In the former procedure, cellular proteins pass into the medium while in the latter method the product is trapped in the bead, which then has to be ruptured to release the product. While this can give high concentrations of antibody, both approaches are limited since cells ultimately rupture the beads and are subject to the limitations of all batch processes.
Continuous culture involves separation of cells from medium on a continuous basis. Several approaches include chemostat cell-retention and cell protecting systems (e.g., hollow fibers, ceramic cartridges). The major advantages of continuous culture over batch cultures are the ability to control the concentration of key medium components; the ability to remove wastes before toxic build-up occurs; the ability to maintain a cell culture in its growth state for greater periods of time; and the ability to reuse medium and expensive growth factors, serum and other additives.
Cell-retention production benefits from the continuous flow of medium which results in higher cell densities and product yield by removing growth inhibitors and supplying a fresh source of nutrients to the cells. Many cell-retention systems utilize microcarriers to grow anchorage-dependent cells. Examples of microcarriers included Bioglas brand hollow glass microspheres (SoloHill, Ann Arbor, Mich.); and charged or collagen-coated microspheres, such as Cytodex brand microcarriers (Pharmacia Fine Chemicals AB). In addition to providing a large surface for attachment, the microcarriers can facilitate suspension and separation. These advantages notwithstanding, the use of microcarriers does not alleviate the aforementioned problem of inadequate oxygen delivery absent vigorous agitation or sparging with gas. As discussed hereinabove, this will generally cause severe cell damage due to the shear forces generated.
Another approach which has been explored extensively in this field is the hollow fiber reactor system wherein medium is perfused through hollow capillary tubes to expose the cells to a more natural environment. Cells can be supported by a constant flow of fresh medium and gases and the products can be removed on-line. One disadvantage with this approach is that the cells grow around and between the capillary tubes and eventually break the fibers. Therefore, this known system cannot be operated for long growth periods, and the cultured cells are difficult to remove.
Another serious disadvantage of hollow fiber reactors is that deleterious metabolic and nutrient gradients develop both axially and radially along the length of the capillary tubes due to the fact that the medium is required to flow quite slowly through the tubes which have narrow inner diameters. Oxygen and carbon dioxide tensions, which are limited by solubility, are maximal at opposite ends of the tubes. The distribution of nutrients, as a result of the slow flow of medium, is effected by diffusion in the hollow fiber reactors. Thus, cells located at different regions in the reactor are subjected to radically different conditions.
It is, therefore, an object of this invention to provide a bioreactor system for mass scale in vitro production of anchorage-dependent and anchorage-independent mammalian cells.
It is also an object of this invention to provide a bioreactor system which will mimic in vivo capillary bed conditions.
It is a further object of the invention to provide a bioreactor system wherein there is independent control of gas concentrations, medium delivery and product removal.
It is an additional object of the invention to provide a bioreactor system wherein cell growth is not limited by oxygen delivery or waste product build-up.
It is still another object of the invention to provide a bioreactor system wherein extensive gas delivery throughout permits advantageous use of lower oxygen concentrations.
It is yet another object of the invention to provide a bioreactor system wherein the problems of shear forces in the growth chamber for oxygenating the cells is obviated.
It is yet an additional object of the invention to provide a bioreactor system wherein negligible pressure and concentration gradients develop along the length of the bioreactor.
It is still an additional object of the invention to provide a bioreactor system wherein movement of nutrients and cell product solutes is achieved by convective flow rather than purely by diffusion.
It is yet a further object of the invention to provide a bioreactor system wherein a multitubular array is resistant to cell-mediated breakdown of the tubes due to excess growth.
It is an additional object of the invention to provide a bioreactor system wherein the individual components can be sterilized, such as by autoclaving.
It is yet an additional object of the invention to provide a bioreactor system which is easy to construct at a relatively low cost and inexpensive to operate.
It is still a further object of the invention to provide a bioreactor system which provides oscillatory flow to ensure that nutrient delivery will be consistent throughout the reactor even at high cell concentrations.
It is yet a further object of the invention to provide a bioreactor system having micro-mixing in the chamber that is similar to a stirred tank reactor, without the deleterious effects of agitation.
It is a further object of the invention to provide a bioreactor system which provides the advantages of a hollow fiber system without the deleterious development of axial and radial gradients.
It is a still further object of the invention to provide a bioreactor system having the capability of permitting sampling of the cell suspension during a production run.